Stem Cell-Based Immune Modulation and Regeneration

Tendinopathies, including both degenerative and inflammatory-based diseases of tendon tissue, include a significant clinical challenge, impacting millions worldwide and imposing a substantial socio-economic burden. Tendinopathies are often chronic conditions induced by an unsolved healing process hesitating in the persistence of active immune cells such as macrophages, neutrophils, and lymphocytes, which release pro-inflammatory cytokines and matrix-degrading enzymes. The sustained inflammatory phase, aggravated by tendons’ inherently low regenerative properties, hinders tissue repair and leads to a poorly organized extracellular matrix, impairing tissue biomechanical properties. Current treatments primarily offer symptom relief without addressing the root inflammatory mechanisms driving the pathology and occurrence of recurrence. This thesis explores the mechanisms behind the emerging pro-tenogenic properties of amniotic epithelial cells (AEC), primarily mediated by their paracrine release of immunomodulatory molecules. The underlying hypothesis proposes that stem cells’ bioactive molecules promote tendon regeneration by facilitating an early shift from the inflammatory phase to the proliferative healing phase. Based on this premise, the thesis aimed to produce AEC immune regulatory secretomes to use them as innovative stem cell-free solutions for controlling the early immune phase of tissue regeneration. The first step assessed whether the immunomodulatory properties of AEC depend on cell phenotype, considering their ability to undergo Epithelial-Mesenchymal Transition (EMT) to support regeneration or interact with different materials. In this context, we found that AEC promptly activated EMT when seeded on graphene oxide (GO)-coated surfaces, a carbon-based nanomaterial that has entered the field with several biomedical applications owing to its exceptional physicochemical and biological features. The cells interacted with GO by activating a specific TGF-β/SMAD signaling pathway that enhances some pro-regenerative AEC properties such as cell migration and adhesion but simultaneously diminishes their ability to inhibit immune cell activation (PBMC), thus confirming that when AEC shift toward the mesenchymal phenotype, their ability to modulate immune responses through paracrine signaling decreases. Subsequently, the study moved towards investigating the molecular mechanism that underlies the paracrine immunomodulatory properties that AEC can express both intrinsically and following exposure to external stimuli. AEC-derived amphiregulin (AREG)/EGFR axis was identified as a key intracellular pathway in regulating immune responses. The activation of this powerful immune-modulatory factor is controlled through the COX-2/PGE2/EP4 axis, which can be activated from different and sometimes converging pathways. First, LPS promotes it through NF-κβ activation but induces YAP degradation, while stretching promotes it through YAP activity. Combined, LPS suppresses stretching's effects, emphasizing the need to balance these stimuli for fine-tuning their anti-inflammatory effects. Beyond cellular responses, this thesis delved into the fractions of the AEC immune modulatory secretome. To this aim, microvesicles (MV) and MV-free fractions were isolated, characterized, and studied for their modulating immune function. It was found that AEC exert their immune modulatory activities using both MV-free and MV secretome fractions. For the first time, we demonstrated that the MV fraction of AEC secretome is enriched of organelle cargo (mitochondria, RE) that can be actively internalized in immune cells, triggering mitochondria-dependent apoptosis and thereby highlighting a potent, cell-free immune suppressive mechanism that could offer therapeutic advantages without the need for direct cell transplantation. Interestingly, the MV-free fraction of the AEC secretome was more effective in inhibiting PBMC and Jurkat cell activation in response to LPS stimuli by rapidly increasing its soluble immunomodulatory components. To translate these findings into potential clinical treatments, the AEC secretome was then encapsulated within a Hyaluronic Acid- Polyethylene Glycol-Heparin (HA-PEG-HEP) hydrogel matrix, which provided a controlled and sustained release of bioactive molecules, significantly bolstering both immunosuppressive and pro-regenerative effects over extended periods. This encapsulation strategy preserved the bioactivity of crucial components like AREG, advancing the therapeutic potential of AEC secretome in a practical, scalable form suitable for clinical use. By harnessing AEC's ability to modulate immune responses and support tissue repair, this dissertation lays the groundwork for the development of cell-free therapies that have broad implications for managing inflammation and degenerative conditions, marking a significant step toward translating AEC-based regenerative therapies from bench to bedside.

Grantor dc:publisher

University of Helsinki

Year dc:date.issued

2025

Author dc:creator

• Cerveró Varona, Adrián

• drug Research

Statement dc:rights

• This publication is copyrighted. You may download, display and print it for Your own personal use. Commercial use is prohibited.
• Julkaisu on tekijänoikeussäännösten alainen. Teosta voi lukea ja tulostaa henkilökohtaista käyttöä varten. Käyttö kaupallisiin tarkoituksiin on kielletty.
• Publikationen är skyddad av upphovsrätten. Den får läsas och skrivas ut för personligt bruk. Användning i kommersiellt syfte är förbjuden.

Language dc:language.iso

eng

Handle dc:identifier.uri

http://hdl.handle.net/10138/591573

OAI record oai:identifier

https://helda.helsinki.fi/server/oai/request?verb=GetRecord&metadataPrefix=dim&identifier=oai%3Ahelda.helsinki.fi%3A10138%2F591573